In this application we intend to test a gene based cell gating mechanism for the isolation of both lineage committed and muttipotent progenitor cells from a heterogeneous population of multipotent stromal cells (MSC). Three specific aims have been identified in order to determine if 1.) a multipotent progenitor cell exists and can be isolated from the stromal reservoir adipose 2.) a lineage committed progenitor for osteoblast differentiation can be isolated from the same heterogeneous cell population 3.) determine if early or late lineage commitment is a reversible process allowing for cellular transdifferentiation. The mechanism for gating cells in this proposal may provide a critical strategy to isolate the necessary cellular building blocks for a number of tissue engineering applications. Given this lab's experience and expertise in studying Distraction Osteogenesis as a model of endogenous bone engineering and remodeling, we have elected to focus on osteoblast differentiation from the MSC compartment. The recent literature suggests a pivotal role for the transcription factor Runx2/Cbfal in the ontogenetic regulation of osteoblast, chondrocyte and possible adipocyte differentiation. Building on the recent literature our strategy is to target the critical early "master gene" Runx2/Cbfat and a late gene indicative of osteoblast commitment osteocalcin (OCN) in order to confer a selectable marker for antibiotic resistance as a cell trap mechanism. If our specific aims are met we have the models and expertise in place to test isolated cells in powerful in vivo models of endogenous bone formation. In addition given the power of this gene trap strategy we will be able to address a fundamental biologic question of stromal cell differentiation, commitment and transdifferentiation from a mixed stromal reservoir. The ability to isolate both progenitor and committed cells is an absolute necessity if regenerative medicine which will encompass cellular therapy and tissue engineering is to be a clinical reality. Our objective is to provide a successful gene based cell trap mechanism for the isolation of progenitor cells to be used as the cellular commodity in the engineering and/or re-engineering of defective connective tissue constructs and joints. This strategy if successful may provide the blueprint for a high stringency cell isolation system that could be utilized for the isolation of numerous other lineage specific cells such as my oblasts and neural progenitors as examples.